The present invention is directed to a method and apparatus for automatically identifying the location of a biological sample in an electrophoresis gel and transferring the sample to a sample plate. More particularly, the invention is directed to a computer assisted method and to a computer controlled apparatus for excising a gel spot from an electrophoresis gel slab and transferring the gel spot to a multiwell sample plate.
Genomes provide the sequence information required to construct proteins that are the working parts of living cells. Genomes and genes are linear constructs composed of four different nucleotides arranged in triplet condons that specify the order and identity of the approximately 20 different amino acids that make up proteins. The nucleic acids are chemically very similar, and are arranged in very long contiguous sequences with intervening non-coding regions. For analysis, nucleic acids must be cut up into fragments of tractable length using shearing forces or restriction enzymes which cut the nucleic acid at specific known sites.
Proteins are made of amino acid subunits that have a range of different isoelectric points, molecular weights, and solubility or hydrophobicity characteristics. The synthesized peptides have exactly defined lengths, and roll up or are assembled into proteins of well defined molecular weights. The estimated 100,000 different primary proteins in man have a range of charge densities and isoelectric points, solubilities, and surface characteristics not found in nucleic acids. Further, proteins have a range of surface conformations which mediate specific interactions between proteins, between proteins and nucleic acids, and, in the form of enzymatically active sites, between low molecular weight metabolites, and all the various types of macromolecules found in cells and foodstuffs. Proteins are the molecular machines that carry out the panoply of syntheses, disassemblies and degradations, immunochemical defense reactions, and paratactic interactions that underlie the assembly of membranes and subcellular organelles.
There is a need for analytical methods that allow a large fraction of the total number of proteins present in a cell or tissue to be detected and quantitated. The quantitative analysis of large sets of proteins that have such a wide variety of functions, sizes, conformation, activities, solubilities, and charge characteristics is both a centrally important challenge, and an exceedingly difficult problem. The problem is rendered even more difficult by the requirement that analysis detecting thousands of proteins per analyses be done in parallel on relatively large numbers of samples in a reasonable time to do experimental toxicological and pharmacological studies.
The electrophoretic mobility of a non-denatured protein is a function of the surface charges of either the monomeric protein or the sum of the surface charges of the subunits, and these are generally used under rate-zonal conditions, i.e., under conditions where the proteins move through a gel or other support at one pH. The distance traveled is a function of the charge to mass ratio, and a function of electrophoresis time. Second dimension separations are done in gradient gels of decreasing pore size such that proteins move until movement essentially ceases as the protein reach pore sizes that prevent further movement. Experimental attempts to develop two dimensional methods based on these parameters using non-denaturing conditions have not yielded the resolution required.
Two-dimensional methods involving denaturing conditions have been explored and widely adopted. The initial separation is done in concentrated urea in the presence of ampholytes which are a heterogeneous mixture of synthetic polymers having wide variation in the ratio of acidic to basic groups. When these are subjected to an electrical field in a gel, the ampholytes sort themselves out into a continuous series based on the isoelectric point. Proteins move along the gel until they reach their own isoelectric point and stop. Further, since the proteins are denatured and unrolled, their isoelectric points reflect the sum of all of the charged groups in the protein, whether previously external or internal in the native state. The isoelectric point determination in such a separation can be calculated from the amino acid composition of the protein, and is a valuable parameter for protein classification.
The second dimensional separation is based on the length (and hence the mass) of the unrolled denatured protein and takes place in the following way. Proteins from the isoelectric separation are exposed to a highly charged detergent which has attached the longest paraffin chain which will remain extended in solution, and not fold back on itself. Sodium dodecyl sulfate (SDS) is the detergent of choice, and in solution will uniformly coat unrolled polypeptide chains, and attach to them by hydrophobic linkages, leaving the highly charged sulfate groups on the surface. The result is particles of approximately rod shape having approximately equal charge-to-mass ratios. Particles having equal charge-to-mass ratios move at the same rate in electrical fields, so that all proteins covered with SDS should have equal mobility in solution. However, if electrophoresis of such particles is done in a microporous gel, then larger particles will be retarded relative to smaller ones.
In practice, the resolutions of these two separate methods are quite high. At least 150 proteins can be resolved from a suitable mixture by isoelectric focusing, and an equal number resolved from a suitable protein mixture by SDS electrophoresis. If the two processes can be mated together in a two-dimensional array, the final resolution should be the product of the resolution of the two methods separately, i.e., 1502 or 22,500. Experimentally, as many as 5,000 proteins have been resolved in large two-dimensional electrophoresis gels, and the theoretical resolution of current electrophoresis as calculated from spot sizes, and the number of spots which could theoretically be packed into the gel area used is around 30,000.
It is quite evident that a key step in the high-resolution two-dimensional electrophoresis technique using isoelectric focusing followed by SDS electrophoresis in the second dimension is mating the two methods together without the loss of resolution inherent in collecting and separately analyzing fractions.
Experimentally, isoelectric focusing is done under temperature controlled conditions in glass tubes (ISO tubes) having an internal diameter of approximately 0.5-2 mm, and approximately 30 cm long. ISO tubes are then attached to a small syringe full of water or buffer solution, and the gels extruded by hand along the top of a second-dimension gel cast between two glass plates. An empty space is typically formed between the top of the gel and the top of the plates. The gels are carefully extruded into this space by a double movement in which the syringe plunger is moved to extrude the gel as the ISO tube containing the gel is moved laterally along the top of the second dimension gel.
The molecules of the test sample migrate through the second dimension gel under the influence of an electric current to isolate the biomolecules. The gel is stained with various stains, such as silver or fluorescent compounds, to visualize the biomolecules. The stained biomolecules are then cut from gel and analyzed by various processes. Typically, the biomolecules are manually cut from the gel slab in the form of a gel spot. The gel spot is then placed in a suitable container. Typically, a single biological sample can be isolated into hundreds of biomolecules that must be manually cut from the gel slab. Accordingly, there is a continuing need in the industry for an improved method and apparatus for cutting samples from a gel slab.
The present invention is directed to an automated apparatus for separating a sample or gel spot from a second dimension electrophoresis gel and transferring the gel spot to a storage container. More particularly, the invention is directed to a computer assisted method and to a computer controlled apparatus for identifying a gel sample containing selected biomolecules in a second dimension electrophoresis gel and transferring the gel sample to a suitable vessel, such as a multiwell sample plate.
Accordingly, a primary aspect of the invention is to provide a method and apparatus for identifying and excising a sample from an electrophoresis gel and transferring the sample to a sample plate.
Another aspect of the invention is to provide a computer controlled robotic arm operatively connected to a computer, where the computer receives an image signal to identify the location of selected samples in an electrophoresis gel and directs the robotic arm to cut the selected samples from the electrophoresis gel.
A further aspect of the invention is to provide an assembly for identifying and separating a sample from an electrophoresis gel, where the assembly includes a remote scanning device and an excising device to excise selected samples from the gel that are identified by the scanning device.
Still another aspect of the invention is to provide a method and apparatus for separating a biological sample from a gel where the gel is supported by a tray that is removably coupled to a gel cutting assembly.
A further aspect of the invention is to provide a method and apparatus for identifying and separating a sample from an electrophoresis gel where the gel is supported on a tray and where the tray can be received in an imaging device to capture an image of the gel and identify selected samples on the gel.
Another aspect of the invention is to provide a tray for supporting an electrophoresis gel where the tray is receivable in an imaging device and receivable in a computer controlled sample cutting device.
Still another aspect of the invention is to provide a tray for supporting an electrophoresis gel where the tray includes a positioning member for positioning the tray in a predetermined location in an imaging device and for positioning the tray in a cutting device for identifying and cutting selected samples from the gel.
A further aspect of the invention is to provide a tray for supporting an electrophoresis gel slab and a carrier device for transporting the gel, where the gel is supported on the tray in a manner to scan an image of the gel and to cut a selected gel sample from the gel slab.
Another aspect of the invention is to provide a tray for supporting an electrophoresis gel where the tray includes at least one aperture for coupling to a computer controlled robotic arm for transporting the tray between work stations.
A further aspect of the invention is to provide a tray for supporting an electrophoresis gel during an imaging process and during a sample cutting process, where the tray includes a first recess for receiving a robotic arm assembly for transporting the tray between work stations and a second recess for cooperating with a robotic arm assembly for loading and unloading a gel onto the tray.
Still another aspect of the invention is to provide a computer controlled assembly for excising a sample from an electrophoresis gel and transferring the sample to a respective well of a multiwell plate where the multiwell plate is delivered from a supply magazine.
Another aspect of the invention is to provide a computer controlled assembly having a conveying apparatus for supplying a sample container from a magazine to an electrophoresis gel cutting device and for conveying the sample container to a storage magazine after receiving a predetermined number of samples.
The various aspects of the invention basically provide an automated apparatus for identifying a sample containing a macromolecule in an electrophoresis gel, excising the sample, and transferring the sample to a multiwell plate. The apparatus includes a computer to control a robotic arm that is able to remove a gel sample or gel spot at a selected location on an electrophoresis gel containing the separated macromolecules. The computer is operatively connected to a plate handling assembly to deliver a multiwell plate from a supply magazine to the cutting apparatus where the samples that are cut from the gel are deposited in a selected well. The sample plate is then transferred to a storage magazine and an empty sample plate is delivered to the cutting apparatus. The computer is connected to an image or scanning device to identify the location of the macromolecules on the gel and to direct the cutting apparatus to a selected location on the gel.
These and other aspects of the invention are basically attained by providing an apparatus for excising a plurality of samples from an electrophoresis gel. The apparatus comprises a base having a work surface with a loading station dimensioned to support a sample plate having a plurality of sample-receiving wells. A gel support member is removably coupled to the base. The support member has a substantially planar surface for supporting the gel. A computer controlled robotic arm has an operating head for excising a plurality of predetermined samples from the gel while being supported on the gel support member and for transferring the predetermined samples to a respective well of the sample plate. A microprocessor is operatively coupled to the robotic arm for controlling movement of the robotic arm. The microprocessor is programmed to receive a signal for identifying the predetermined samples on the gel, actuating the robotic arm to excise the predetermined samples, and transferring the samples to the respective well of the sample plate.
The aspects of the invention are further attained by providing an apparatus for excising a plurality of biological samples from an electrophoresis gel. The apparatus comprises a base having a work surface with a loading station dimensioned to support an electrophoresis gel and a sample plate having a plurality of sample-receiving wells. An automated sample plate handling assembly is coupled to the base. The plate handling assembly includes a supply magazine for containing a plurality of sample plates, a receiving magazine, and a conveyor for sequentially conveying a sample plate from the supply magazine to the loading station and for conveying the sample plate from the loading station to the receiving magazine. A robotic arm has an operating head for excising a plurality of predetermined samples from the gel and for transferring the excised samples to a respective well of a sample plate positioned in the loading station. A microprocessor is operatively connected to the robotic arm for controlling movement of the robotic arm. The microprocessor is programmed to receive a signal for identifying the predetermined samples on the gel and actuating the robotic arm to excise the respective predetermined samples and transferring the predetermined samples to the respective well of the sample plate.
The aspects of the invention are also attained by providing a computer assisted method for transferring a biological sample from an electrophoresis gel in a cutting assembly to a respective well of a sample plate. A sample plate is delivered from a supply magazine to a sample loading station, where the sample plate includes a plurality of spaced-apart sample wells. A positioning signal is produced indicating the presence of a sample plate in the loading station and transmitting the positioning signal to a computer. An excising signal is produced in the computer and directs a robotic assembly of the cutting assembly in response to the excising signal to excise a biological sample from the electrophoresis gel and transfer the sample to a predetermined well of the sample plate.
The aspects of the invention are still further attained by providing a method of excising and transferring a biological sample from an electrophoresis gel to a respective well of a sample plate. The method comprises the steps of: providing a gel excising apparatus having a top surface for supporting an electrophoresis gel, a loading station dimensioned for supporting a sample plate, a robotic arm having an operating head and being operatively connected to a microprocessor, a supply magazine having a plurality of multiwell sample plates; supplying a sample plate from the supply magazine to the loading station; positioning an electrophoresis gel on the top surface; actuating the robotic arm to excise a predetermined sample from the gel and transferring the excised sample from the gel to a respective well of the sample plate.
The various aspects, advantages and other salient features of the invention will become apparent to one skilled in the art in view of the following detailed description of the invention and the annexed drawings which form a part of this original disclosure.